The present invention relates to a method of purifying a set of specific DNA molecules to be used in DNA-DNA hybridisations, as well as to DNA probes containing less than 2% Cot-1 DNA.
Molecular genetics is the study of nucleic acids and their role in the biology of the cell. At the core of this science is the technique of Southern blotting, which involves the hybridisation of DNA in solution to DNA immobilised on a solid membrane. One relatively new branch of molecular genetics, molecular cytogenetics, deals with the molecular biology of the chromosome level of organisation as opposed to the DNA level. The field of molecular cytogenetics is a steadily expanding field whose broad implications for the study of human and other genomes have not yet been fully explored. Examples of the kind of experiments carried out within this field are given below.
Fluorescence in situ hybridisation (FISH) has been applied with a multitude of probes of different complexity for chromosome painting (Lichter et al., 1988;
Pinkel et al., 1988) and chromosome bar coding (Lengauer et al., 1993) and has provided the most direct and rapid way to map the chromosomal localisation of DNA sequences (Lichter et al., 1990). FISH to extended chromatin fibres and single DNA strands has brought the mapping resolution down to the kilobase (kb) range offering powerful new possibilities for the generation of high resolution physical maps (Florijn et al., 1995, Weier et al., 1995).
Multicolour FISH approaches taking advantage of the combinatorial use of six fluorochromes have allowed to distinguish each of the 24 human chromosomes by a different colour (Speicher et al.; 1996, Schrock et al., 1996). FISH to extended DNA molecules has brought down the mapping resolution of this approach to the kb range offering powerful new possibilities for the generation of high resolution physical maps (Weier et al., 1995; Florijn et al., 1995).
Interphase FISH (xe2x80x9cinterphase cytogeneticsxe2x80x9d; Cremer et al., 1986) has allowed the study of numerical and structural chromosome aberrations directly in the cell nucleus.
Comparative genomic hybridisation (CGH, Kallioniemi et al., 1992, du Manoir et al., 1993; Joos et al., 1993) has provided a powerful tool to detect non-random gains and losses of DNA sequences in genomic DNA (obtained, for example from tumour specimens).
Procedures for the quantitative and automated evaluation of FISH experiments have been developed in parallel and hold the promise for fully automated optical mapping approaches in the future.
Present diagnostic and research applications range from prenatal diagnosis to postnatal clinical cytogenetics, from studies of genetic changes in cancer (du Manoir, 1995; Piper et al., 1995) to biological dosimetry (Cremer et al., 1990; Lucas et al., 1992), from comparative chromosome mapping (Wienberg et al., 1990; 1995) to studies of the 3D-organisation of genomes in situ (Manuelidis, 1990; Cremer et al., 1993). Chromosome-specific cDNA and other libraries, as well as subregional probes, for example microdissection probes, YACs, BACs, PACs, cosmids that are presently available, often do not optimally serve the needs of molecular cytogenetics applications. Several examples may serve to demonstrate the needs of improved probes:
All probes presently available for Southern blotting and FISH which are derived, for example, from cosmid, BAC, PAC and YAC libraries contain interspersed repetitive sequences. This presents a problem when probes derived from genomic DNA are hybridised to DNA on Southern blots or to chromosomes/nuclei in situ, since the interspersed repetitive sequences present in the probe DNA hybridise throughout the target DNA, i.e. to all of the DNA present on Southern blot or within all chromosomes/nuclei in situ. Thus the hybridisation to the true target sequence is obscured by background hybridisation everywhere else. To prevent non-specific hybridisation between interspersed repeat sequences of probe and target, an excess of unlabeled competitor DNA is usually included in the hybridisation mix as xe2x80x9cblockingxe2x80x9d agent. Southern blotting techniques usually use total human DNA for this purpose (Sealey et al, 1985) and FISH, Cot-1 DNA (Pinkel et al., 1988; Lichter et al., 1988). Cot-1 DNA is highly enriched for sequences present more than 104 copies per haploid genome. However, the routine inclusion of commercial sources of Cot-1 DNA in hybridisation mixtures in excess quantities is expensive. A certain fraction of labelled, interspersed sequences will hybridise to target sequences even in the presence of excess Cot-1 DNA and lower the signal to background ratio of hybridisation signals. Thus there is an urgent need for the development of improved probes which entirely lack repetitive sequences which are shared with other chromosomes and thus impair the specificity of the probes.
In studies employing GGH to chromosomes or to DNA microarrays (Kallioniemi et al., 1992; du Manoir et al., 1993; Schena et al., 1995; Shalon et al., 1996), representational difference analysis (RDA; Lisitsyn et al., 1993) or genomic mismatch scanning (Nelson et al., 1993), it would be clearly advantageous if the DNA used for such studies would not comprise the entire complexity of a large genome, but a representative sample highly enriched in single copy or coding sequences,
In multicolour FISH studies employing combinatorial probe labelling (Speicher et al.; 1996, Schrxc3x6ck et al., 1996), it would be advantageous if probe sets have no repetitive sequences. Usually, so many different probes have to have be hybridised a with correspondingly large amount of Cot-1 DNA, thus making hybridisations both expensive, bulky and liable to have low signal to background ratios.
In FISH studies of chromosome evolution, probes representing entire genomes, as well as chromosome or chromosomal subregions need to be enriched for sequences conserved between two species of interest to define more readily evolutionary conserved segments along chromosomes, as well as evolutionary chromosomal rearrangements in species belonging to a given class or even to different classes.
In studies of 3D in situ human genome organisation, chromosome- and chromosome region-specific paint probes containing specific subsets of sequences would be highly useful, such as complementary sets comprising coding sequences vs. non-coding sequences, xe2x80x9cscaffoldxe2x80x9d attached sequences vs. non-attached sequences.
If a DNA probe is generated by PCR or is present within a vector, the knowledge of whose sequence facilitates PCR amplification, then the probe can be further amplified using the existing primers. Some complex probe sets are amplified using a universal PCR amplification protocol. This means that when the probe set is first selected, usually from an amount of DNA corresponding from a small number of nuclei, it is amplified in a way that maximises amplification of all DNA fragments. There are at least two ways of doing this: DOP-PCR and linker-adapter PCR.
Since 1992, universal DNA amplification procedures have been introduced that allow the amplification of any DNA sources employing primers which contain a stretch of random base pairs and another stretch with a specific DNA sequence (Telenius et al., 1992, Bohlander 1992). The method described by Telenius et al., (1992) termed degenerate oligonucleotide-primed (DOP)-PCR is well established in our laboratories, with the same oligonuctide [SEQ ID NO.: 1] (xe2x80x9c6MWxe2x80x9d, 5xe2x80x2-CCG ACT CGA GNN NNN NAT GTG G-3xe2x80x2) and conditions as Telenius et al., (1992). During the first five PCR cycles, which are performed under conditions of lower stringency (i.e. a low annealing temperature), the primer part comprising the random (N6) sequence can hybridise to many sites of any complex DNA source. Subsequent cycles performed under stringent conditions (i.e. a higher annealing temperature) should, in theory, allow the specific, further amplification of those DNA fragments in which the specific primer sequence has been incorporated during the first amplification cycles.
Linker-adapter PCR strategies involve the generation of specific target sequences at the end of DNA fragments which hybridise to unique primer pairs. This approach allows the amplification and reamplification of complex sources of DNA such as DNA from microdissected chromosomes, chromosome arms and chromosome bands (Lxc3xcdecke et al., 1989; Vooijs et al., 1993).
Subtractive hybridisation involves the hybridisation of two sets of DNA molecules, the source DNA and the subtractor DNA. Usually, the contents of the these two DNA sets overlap. The relative amounts of source and subtractor DNA can be altered to drive the hybridisation kinetics to favour either positive or negative selection. Positive selection, in which the number of source molecules is usually greater, would involve isolation of subtractor-homologous source DNA. Negative selection, in which the number of subtractor molecules is greater, would involve isolation of subtractor-nonhomologous source DNA. In addition, the selected DNA can be put through more round(s) of selection to further improve the selection process. Subtractive hybridisation was first used to clone the differences between two overlapping pools of cDNAs; double-stranded hybrid molecules were separated from free single-stranded cDNAs by chromatography through hydroxylapatite (Kurtz and Feigelson, 1977; Affara and Daubas, 1979; Timberlake, 1980). This enabled only negative selection. With the substitution of biotin-labelling of the subtractor DNA followed by avidin-affinity capture and release techniques, it became possible to both positively and negatively select subsets of source DNA (Welcher et al., 1986). This technique was further improved by the addition of a post-selection inter-Alu-PCR amplification step (Rounds et al., 1995).
The concept of removing repetitive sequences from chromosome-specific libraries employed for the FISH-visualisation of entire chromosomes and chromosomal subregions by affinity chromatography procedures and alternatively to suppress the undesired hybridisation of hapten-labelled repetitive probe sequences with Cot-DNA fractions as blocking agents was first tested by T. Cremer and P. Lichter when they worked together with David C. Ward at the Dept. of Genetics, Yale University (1986-1988). Copper chelation affinity chromatography, a well known procedure for the purification of proteins, had already been modified there for use in the enrichment of specific DNA sequences from complex DNA sources (Welcher et al., 1986).
The following strategy to produce a human chromosome 21 xe2x80x9cpaintxe2x80x9d probe library depleted in interspersed repetitive sequences, as well as other repetitive sequences cross-hybridising to other acrocentric chromosomes was tested at that time. In the following strategy, source DNA is represented by a chromosome 21 paint probe and the subtractor by biotinylated and 3H-labelled sequences representing flow sorted chromosomes 13 and 18, and cloned repetitive sequences. These subtractor sequences were used in excess (50 fold) and mixed with chromosome 21 subtractor sequences end-labelled with 32P. The radioactive labelling was performed as a simple means to follow the presence of subtractor and source sequences and the intended fractionation of the latter. Following hybridisation of subtractor and source DNA in suspension, and incubation with avidin, the hybridisation mixture was subjected to copper chelation chromatography. The affinity of the columns for avidin was used with the intention of removing all biotinylated subtractor DNA sequences and subtractor DNA-source DNA hybrids (3H labelling was used to monitor the bound fraction and possible leakage of the column). The expected enrichment of chromosome 21-specific, 32P-labelled source sequences in the flow-through fraction (negative selection) was monitored by a filter hybridisation assay with spots representing the various components of the subtractor DNA and the chromosome 21 source DNA. After several cycles, a strong increase in the intensity of the chromosome 21 spot demonstrated the enrichment of chromosome 21-specific sequences in the flow-through fraction.
However, a major drawback of this approach resulted from the fact that the columns retained the large majority but less than 100% of the biotinylated subtractor DNA. This means that the fraction of excess biotinylated subtractor sequences recovered in the flow-through fraction reflected a total amount similar or even higher than the amount of the purified source DNA. This fraction of subtractor DNA which leaked into the flow through fraction made the total DNA recovered in the flow through fraction at this stage of the development useless as a probe for immediate molecular cytogenetic applications, in spite of the experimentally-demonstrated enrichment of chromosome 21 unique sequences. As a result of this leakage, repetitive sequences, which were depleted from the 21 library probe, were replaced in the flow-through fraction to a significant extent by repetitive sequences from the subtractor DNA. Accordingly, this attempt to prepare a 21-specific chromosome paint probe which could be used without Cot-1 DNA in the hybridisation mixture failed, i.e. FISH experiments of the resulting probes to metaphase spreads did not yield a specific painting of chromosome 21, but still visualised the entire chromosome complement. In the case of positive selection (i.e. subsequent elution of a fraction bound to the column) the situation is even worse, since all subtractor DNA bound to the column will be eluted together with the subtractor-source DNA hybrids, using this approach.
Two recent publications have used two modifications to the previously-used subtractive hybridisation protocol to positively select chromosome-specific cDNAs (Chen-Liu et al., 1995; Rouquier et al., 1995). Firstly, source cDNA libraries were made universally amplifiable by the addition of linker-adapter ends. Using single chromosome libraries as subtractor DNA, chromosome-specific cDNAs were PCR-amplified after positive selection. A further step was achieved by using streptavidin-coated magnetic beads for affinity chromatography, hence reaction volumes were kept to a minimum. In addition, single-stranded source molecules could be released from immobilised subtractor molecules by alkaline denaturation after stringent washes, thus further improving the purity of the released source DNA. When two consecutive rounds of positive selection were employed, this led to a selection of chromosome-specific cDNA. However, the method described in these two publications have used Cot-1 DNA to suppress any repeat-repeat hybridisation within or between subtractor and source DNA (Chen-Liu et al., 1995; Rouquier et al., 1995). This makes the process of positive selection more complicated and makes negative selection almost impossible (the unlabelled Cot-1 DNA would pass through into and heavily contaminate the supernatant to be amplified for negative selection).
Thus, the technical problem underlying the present invention is to provide a novel and generally useful strategy to fractionate and purify DNA from entire genomes, as well as from chromosome-specific or chromosome segment-specific DNA probes. This approach can be used to develop probe sets for molecular cytogenetics with novel and different characteristics, such as probe sets comprising coding sequences, expressed sequences, sequences conserved between two distantly-related species, etc. Such probe sets will be generated in a way that they can be reamplified and used without any additional xe2x80x9cblockingxe2x80x9d agents such as Cot-1 DNA. These novel probe sets will become highly useful in diagnostic and research settings.
The above technical problem is achieved by providing the embodiments characterized in the claims.
In particular, there is provided a method of purifying specific DNA molecules, comprising the steps of
(a) mixing a set of DNA molecules as source DNA containing said specific DNA molecules as a subset, with a set of molecules as subtractor having substantially
(i) an affinity for the subset, or
(ii) no affinity for the subset,
(b) performing a binding reaction between source DNA and subtractor in solution,
(c) separating the subtractor which is present unbound or bound to source DNA, by binding the subtractor to a matrix material containing compounds having an affinity to the subtractor, from the reaction mixture, and
(d) recovering said subset which is either
(i) bound to the subtractor, or
(ii) not bound to the subtractor.
In the case of the feature of step (a) (ii), the subtractor having substantially no affinity for the subset, exhibits an affinity for substantially all other DNA molecules contained in the source DNA.
The recovered subset obtained in step (d) (i) derives from a xe2x80x9cpositive selectionxe2x80x9d using preferably an excess of source DNA. The recovered subset obtained in step (d) (ii) derives from a xe2x80x9cnegative selectionxe2x80x9d using preferably an excess of subtractor.
In a preferred embodiment, the subtractor is a set of DNA molecules and the binding reaction in step (b) is a hybridisation. In this aspect, the hybridisation may be performed by denaturing source DNA and subtractor mixed together in a buffer containing 0.075 M to 1.5 M NaCl, at 90 to 100xc2x0 C. for 1 to 10 minutes and reannealing at 60 to 70xc2x0 C. for 5 to 48 hours. Alternatively, the hybridisation may be performed by denaturing source DNA and subtractor mixed together in a buffer containing 40 to 70% v/v formamide, 0.03 M to 0.75 M NaCl, at 60 to 80xc2x0 C. for 1 to 10 minutes and reannealing at 30 to 50xc2x0 C. for 5 to 48 hours.
In a further preferred embodiment of the present invention, the source DNA is a DNA probe used in FISH. The source DNA may comprise DNA of a defined function or a repeat-free chromosane-specific library. Examples are cDNA, CpG islands, scaffold-attached DNA and DNA of a defined replication time.
The subtractor may be a set of labelled molecules such as DNA molecules, wherein the percentage of labelling is xe2x89xa790%, more preferred xe2x89xa795%, and most preferred xe2x89xa797% such 99.5%. The molecules are labelled e.g. with biotin or digoxygenin, wherein the label or labelling method are not specifically restricted and any label or labelling method known in the art can be used within the present invention. In a preferred embodiment of the present invention, the subtractor is Cot-1 DNA.
Cot DNA means a double stranded DNA formed after a mixture of DNA molecules, at a known concentration of DNA and salt, which has been denatured and reannealed until a certain value of the product of molar concentration (Co) and time (t) has been reached. Cot-1 DNA represents the double stranded DNA formed after DNA has been denatured and reannealed until the product of molar concentration and time is 1, and represents sequences repeated more than 10,000 times per haploid human genome.
The subtractor may comprise a repeat-free chromosome-specific library or DNA of a defined function. Examples are cDNA, CpG islands, and scaffold-attached DNA.
Preferred embodiments of the present invention with respect to source DNA and subtractor are as follows:
the source DNA comprises repeat-free DNA from one species and the subtractor comprises repeat-free DNA from another species;
the source DNA comprises repeat-free DNA from one species or DNA sequences shared between two different species, and the subtractor is a repeat-free chromosome-specific library from another species;
the source DNA is a repeat-free chromosome-specific library from one species, and the subtractor comprises repeat-free DNA from another species or DNA sequences shared between two different species;
the source DNA comprises repeat-free DNA from one particular tissue and/or developmental stage, and the subtractor comprises repeat-free DNA of a defined function from another particular tissue and/or developmental stage of the same organism.
The matrix material used in step (c) is not specifically restricted, and any matrix material known in the art can be used within the present invention. The compounds contained in the matrix material are preferably immobilized to the matrix material. In a preferred example, the matrix material contains streptavidin such as streptavidin-conjugated magnetic beads.
In another embodiment of the present invention, the source DNA contains thymidine analogues, the subtractor contains anti-thymidine analogue antibodies, and the matrix material contains compounds capable of binding to the anti-thymidine analogue antibodies. Alternatively, the source DNA contains bromode-oxyuridine (BrdU), the subtractor contains anti-BrdU antibodies, and the matrix material contains compounds capable of binding to the anti-BrdU antibodies. Prior to step (a) of the method according to the present invention, the source DNA may be subjected to PCR such as DOP-PCR. In a preferred embodiment, prior to step (a), the source DNA is subjected to a further round of PCR with a second primer whose 5xe2x80x2-portion comprises a sequence of nucleotides not present in the DOP primer, and whose 3xe2x80x2-portion consists of a number of the non-random nucleotides from the 5xe2x80x2-end of the DOP-prer, Preferably, the DOP-PCR primer (xe2x80x9cfirst primerxe2x80x9d) has the following sequence [SEQ ID NO.: 1]:
5xe2x80x2-CCG ACT CGA GNN NNN NAT GTG G-3xe2x80x2
wherein N may be any nucleotide.
Further, the second primer may have the following sequences [SEQ ID NOS: 2-3]:
5xe2x80x2-CTA CTA CTA CTA CCG ACT CGA G-3xe2x80x2, or
5xe2x80x2-TGA TCA CGC TAC CCG ACT CGA G-3xe2x80x2.
After step (d) of the method according to the present invention, the recovered subset may be subjected to PCR. Further, steps (a) to (d) are repeated at least once using each recovered subset obtained in step (d) as source DNA in step (a).
The subset obtained according to the method of the present invention may be used as a probe for DNA-DNA hybridisations such as Southern blotting or FISH.
A further subject of the present invention relates to a DNA probe containing less than 2% Cot-1 DNA.
The source DNA are preferably selected from probes cloned in vectors, complex probe sets created by PCR amplification and/or microdissection or flow-sorting, repeat-free chromosome-specific library, repeat-free genomic DNA purified from cells with tymidine analogues incorporated into R- or G-bands, early or late replicating genomic DNA and repeat-free genomic DNA. The source DNA originates from any living material such as plants, fungi, bacteria, animals and humans, and specific tissues or cells thereof. Examples are: Humans, mice, sheep, cows, horses, pigs, goats, rabbits, ostriches, chickens, pigeons, maize, wheat, rice, barley, oats, rye, sorghum, millet, and yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe.